Description (applicant's description): The long-term goal of the project is to understand the structural basis of signaling mediated by the TGF-beta cytokines. TGF-betas play important and diverse roles in mammalian endocrine function. In particular, their ability to induce growth arrest and promote extracellular matrix formation has direct relevance to the understanding of uncontrolled tumor proliferation in cancer and renal hypertrophy in diabetic nephropathy. TGF-betas signal through type I and type II transmembrane receptor serine/threonine kinases. Ligand binding induces type I-type II receptor association, resulting in phosphorylation and activation of the type I kinase by the type II kinase. The signal is then transduced by cytoplasmic smad3, which is activated by receptor kinase-mediated phosphorylation. Phosphorylated smad3 forms a heteromeric complex with smad4, and the complex enters the nucleus to regulate transcription. This proposal examines the molecular mechanism of phosphorylation-induced smad3-smad4 association. Preliminary studies have demonstrated a direct role of smad3 C-terminal pseudophosphorylation in promoting smad3-smad4 interaction in vitro. The specific aims determine the structural basis of the interaction using biochemical and biophysical approaches. Site-directed mutagenesis will be employed to map subunit interacting surface and the binding site for the phosphorylated C-terminal tail of smad3. A semi-synthetic approach using intein-mediated protein ligation to generate the phosphorylated form of smad3 will be used to address the effect of physiological phosphorylation on heteromeric interaction. Chemical cross-linking experiments will be used to probe subunit stoichiometry within the hetero-oligomer and provide subunit-to-subunit distance information. Sedimentation velocity and equilibrium experiments will be performed to investigate the association model and the energetic of heteromeric association. X-ray crystallography will be used to determine the detailed molecular interaction. The approaches are complementary and will link existing biological information on SMAD protein function to the detailed structural basis of action. Elucidating the structural basis for these signaling interactions would increase our understanding of these processes and thereby ability to generate effective therapeutic reagents for human diseases.